Abstract
The hepatitis E virus causes acute viral hepatitis endemic in much of the developing world and is a serious public health problem. However, due to the lack of an in vitro culture system or a small animal model, its biology and pathogenesis are poorly understood. We have shown earlier that the ORF3 protein (pORF3) of hepatitis E virus activates ERK, a member of the MAPK superfamily. Here we have explored the mechanism of pORF3-mediated ERK activation and demonstrated it to be independent of the Raf/MEK pathway. Using biochemical assays, yeast two-hybrid analysis, and intracellular fluorescence resonance energy transfer we showed that pORF3 binds Pyst1, a prototypic member of the ERK-specific MAPK phosphatase. The binding regions in the two proteins were mapped to the N terminus of pORF3 and a central portion of Pyst1. Expression of pORF3 protected ERK from the inhibitory effects of ectopically expressed Pyst1. This is the first example of a viral protein regulating ERK activation by inhibition of its cognate dual specificity phosphatase.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Blotting, Western
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COS Cells
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Cell Cycle Proteins*
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Cell Line
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Cytoplasm / enzymology
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Dual Specificity Phosphatase 1
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Dual Specificity Phosphatase 6
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Enzyme Activation
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Enzyme Inhibitors / pharmacology
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Fluorescence Resonance Energy Transfer
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Glutathione Transferase / metabolism
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Immediate-Early Proteins / antagonists & inhibitors*
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Immediate-Early Proteins / metabolism*
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JNK Mitogen-Activated Protein Kinases*
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MAP Kinase Kinase 4
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Mitogen-Activated Protein Kinases / metabolism*
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Models, Biological
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Open Reading Frames
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Phosphoprotein Phosphatases*
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Phosphoric Monoester Hydrolases / metabolism
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Plasmids / metabolism
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Precipitin Tests
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Protein Binding
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Protein Biosynthesis
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Protein Phosphatase 1
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Protein Structure, Tertiary
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Protein Tyrosine Phosphatases / antagonists & inhibitors*
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Protein Tyrosine Phosphatases / metabolism*
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Signal Transduction
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Time Factors
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Transfection
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Two-Hybrid System Techniques
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Viral Proteins / metabolism*
Substances
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Cell Cycle Proteins
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Enzyme Inhibitors
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Immediate-Early Proteins
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ORF3 protein, Hepatitis E virus
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Viral Proteins
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Glutathione Transferase
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 4
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Mitogen-Activated Protein Kinase Kinases
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Phosphoprotein Phosphatases
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Protein Phosphatase 1
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Phosphoric Monoester Hydrolases
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Dual Specificity Phosphatase 1
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Dual Specificity Phosphatase 6
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Protein Tyrosine Phosphatases